One of the main challenges facing today's tomato industry is how to deliver to a processing plant or to the marketplace tomato fruit that have been vine-ripened (and thus are desirable to consumers in terms of taste, texture, and color), but that remain firm without the usual post-harvest ripening-related softening that reduces shelf life of harvested fruit. Using traditional breeding methods, which are very labor intensive, it could take years to develop a novel tomato variety that ultimately may display only a modest increase in shelf life. Instead, recent studies have utilized genetic and biochemical techniques in an effort to identify the factors that regulate fruit ripening. By identifying and modifying the expression of specific genes, researchers and breeders hope to develop new tomato varieties that have the desirable qualities of vine-ripened fruit, but that are resistant to post-harvest softening and therefore display an extended shelf life.
Fruit softening is one of the many ripening-related changes—including alterations in fruit texture, color, aroma, and metabolism of sugars and organic acids—that occur as a result of a developmental program triggered by ethylene. The changes associated with ripening, in particular post-harvest softening, limit the shelf life of fresh produce, such as tomatoes. Several genes associated with the ripening process in tomato have been identified and include at least seven members of a family of genes called the β-galactosidase genes (Smith and Gross, Plant Physiology 123:1173-1184, 2000).
β-galactosidases comprise a family of genes that catalyze the hydrolysis of terminal galactosyl residues from carbohydrates, glycoproteins, and galactolipids. One family member, tomato β-galactosidase 4 (TBG4), codes for the enzyme β-galactosidase II, which has been proposed to play a role in cell wall degradation that underlies fruit softening.
Consistent with this idea, antisense down-regulation of TBG4 was reported to increase tomato fruit firmness by up to 40% compared to control fruit (Smith et al., Journal of Experimental Botany 54(390):2025-2033, 2002). Though the authors concluded that the “presence of the TBG4 antisense construct is linked to significantly firmer fruit in four of the six antisense lines” they went on to state that “there are no clear correlations linking the biochemical data to the increased firmness among all the antisense lines when compared with control.” Further, data presented by Smith et al. in FIGS. 1 and 2 fail to confirm the efficacy of their TBG4 antisense construct. Not only were the authors unable to replicate the suppression of TBG4 mRNA levels that they first observed in fruit from line 1-1 at breaker plus 3 days (B3) (see FIG. 1) in a second study (FIG. 2), they reported that TBG4 mRNA levels were unexpectedly more abundant in fruit from four antisense lines than in parental control fruit at breaker plus 7 days (B7). These expression data show that the antisense construct did not constitutively suppress TBG4 expression as the authors expected. Because TBG4 cDNA shares approximately 70% nucleotide sequence identity to other β-galactosidase gene family members, Smith et al. examined expression of several TBG genes in fruit from line 1-1 to evaluate the specificity of their antisense suppression. Compared to parental control fruit, TBG3 mRNA levels in fruit from line 1-1 were significantly lower at mature green and significantly higher at B3 and B7. Taken together, these observations raise the possibility that the alterations Smith et al. observed in fruit firmness were not the result of antisense suppression of TBG4 mRNA expression. Because there are no characterized mutations of the TBG4 gene in tomato, the role of this gene in fruit firmness has not been assessed using an independent approach that specifically targets the TBG4 gene. The method described herein, in contrast to antisense technology, can be used to specifically target the TBG4 gene despite its high identity with other family members.
Transgenic approaches targeting the TBG4 gene have been proposed to modify β-galactosidase gene expression and β-galactosidase II protein expression during tomato fruit development (U.S. Pat. No. 6,872,813; U.S. Patent Publication No. 20050014267 A1). However, public acceptance of genetically modified plants, particularly with respect to plants used for food, is not universal. Since many consumers have clear preferences against genetically modified foods, it would be useful to have a tomato exhibiting reduced levels of TBG4 that was not the result of genetic engineering methods. A cultivated tomato that is firmer when ripe and has reduced post-harvest fruit softening as a result of altered TBG4 protein that is not the result of genetic engineering would have tremendous value for the tomato industry, including fresh market and processor tomatoes. Such a tomato could be used in a variety of tomato food products for example, sliced tomatoes, canned tomatoes, ketchups, soups, sauces, juices and pastes.
To date, mutations in the TBG4 gene of tomato have not been reported and no one has reported or described a naturally occurring “knockout” or “knockdown” of TBG4. Therefore, the effect of “knockout” or “knockdown” of TBG4 on tomato fruit firmness is not known. It would be useful to have an allelic series of mutations in the TBG4 gene that provide a spectrum of phenotypes that could be used to optimize the breeding tomato varieties that retain many of the quality traits of vine-ripened tomatoes, yet have an extended shelf life. Tomato lines with TBG4 mutations that have been genetically characterized could also be crossed with lines that carry mutations in other genes involved in ripening. A cultivated tomato that is firmer and has reduced post-harvest softening as a result of its TBG4 gene being either knocked out or otherwise hindered that is not the result of genetic engineering would not only confirm the importance of this particular β-galactosidase gene in tomato fruit softening, but would have tremendous value for the entire tomato industry.